Chromatographic processes generally effect separation of a mixture into its components by dissolving the mixture in a mobile phase and passing the mobile phase over a stationary phase. Separation is achieved by differential partitioning of the components of the mixture between the mobile phase and the stationary phase. A mixture of analytes can be separated chromatographically into discrete subpopulations by utilizing intrinsic properties of the analytes, such as polarity, charge, function, and size.
Various chromatographic methods are known for size-based analyte separation. The most common method utilizes a steel column packed with a stationary phase consisting of spherical particles of silica or a polymer, with diameters typically in the range from approximately 4 to 17 microns. The spherical particles have pores of varying sizes. When the mobile phase passes over the particulate stationary phase, the entry of analytes into the pores depends on the sizes of the analytes as well as on the sizes of the pores. Smaller analytes enter more pores than larger analytes, and therefore tend to reside for a longer time within the column. Consequently, the concentration of larger analytes in the effluent of the column tends to be greatest at the beginning of the process while the concentration of smaller analytes in the effluent tends to become greatest later on in the process.
Size-based chromatographic methods include size-exclusion chromatography (SEC), gel filtration chromatography (GFC), and gel permeation chromatography (GPC). Although these methods are widely used, they are not without shortcomings.
A first drawback is that the particulate stationary phase in a chromatographic column can act as a filter. Some large analytes may not pass through the column at all, and therefore go undetected.
A second drawback is that separations utilizing chromatographic columns tend to be slow. Separation and column re-equilibration often take 60 minutes or more. In theory, separation time can be decreased by increasing the flow rate of the mobile phase. However, at best, the separation time can be decreased only by a modest amount because increasing the flow rate increases the pressure on the stationary phase and the increased pressure can crush the porous particles, destroying the column's capacity for separation.
A third drawback is that the particles in a silica-based stationary phase begin to dissolve when exposed to a liquid having an alkaline pH (i.e., a pH greater than 8). On the other hand, although polymeric-based stationary phases can readily accommodate a greater pH range, some analytes can be absorbed by polymeric-based particles. The mutual affinity for some analytes and polymeric particles introduces an additional differentiating property into the separation process, and the process becomes a mixed mode process, no longer based exclusively on the size of the analyte.
Capillary Hydrodynamic Fractionation (CHF) is another sized-based separation technique. In CHF, liquid flows through a long, narrow capillary tube. The velocity profile of the flow through the capillary is approximately parabolic (Poiseuille flow). The velocity of the liquid is greatest at the center of the capillary, and decreases toward the capillary wall, where it becomes almost stagnant. Analytes injected into the capillary spread through the capillary's bore and experience all possible flow rates. At any given moment, an analyte particle migrates at the flow rate closest to its hydrodynamic center. The closest approach of the hydrodynamic center of an analyte particle to the capillary wall is approximately equal to the particle's hydrodynamic radius. Smaller analytes can therefore approach the capillary wall more closely than the larger analytes, and their movement, when closer to the capillary wall, is influenced by the lower liquid flow rates adjacent the capillary wall. The result is that larger analytes remain in the vicinity of the higher flow rates in the capillary and have a higher mean velocity than smaller analytes. The larger analytes are therefore eluted first, and separated from smaller analytes.
The absence of a particulate stationary phase in CHF is advantageous because fast separation times (e.g., 7-10 minutes) can be achieved. However, the CHF technique has poor resolution and is primarily applicable in the separation of colloids. In addition, the very narrow capillary bore (typically having a diameter in the range from approximately 1 to approximately 10 microns) makes detection of the analytes difficult.
CHF has not found significant application in the separation of biological molecules such as proteins, which often have sizes less than the typical separation range of approximately 0.015 to 1 micron in CHF.
Hydrodynamic chromatography (HDC) is a size-based separation technique that includes elements of both GPC and CHF. HDC utilizes a tubular column containing a particulate stationary phase as in GPC. However, unlike the stationary phase particles in GPC, the particulate stationary phase in HDC is non-porous. The nonporous particles form a network of interstitial spaces accessible by the liquid mobile phase. The interstitial spaces are intended to approximate the long capillary tube found in CHF. As in CHF, analytes are separated in HDC on the basis of their mean velocity due to the Poiseuille flow along the walls of the column and along the surfaces of the nonporous particles that form the interstitial spaces.
The typical separation range for HDC is approximately 0.01-10 microns, and has limited the technique mostly to the analysis of submicron colloidal particles. HDC suffers from many of the same shortcomings as GPC, including long analysis times and potential column damage due to high pressures.
Flow field-flow fractionation (FFF) is a sizing technique applicable to both biological molecules (e.g., proteins) and colloids. The fractionation device consists of a separation channel approximately 10-50 centimeters long, 1-3 centimeters wide and 0.01-0.05 centimeters thick. The channel is bounded by an upper wall and a lower wall, the latter referred to as an “accumulation wall.” The upper wall is formed by a permeable membrane, and the lower wall is porous and covered by a filter that serves as a barrier to analytes. During the separation process, liquid flows lengthwise in the channel in laminar flow, with a parabolic velocity profile similar to that found in CHF. Simultaneously, flow perpendicular to the length of the channel emanates from the membrane forming the upper wall, creating a convective flux. Liquid exits the channel both through the accumulation wall, and through the distal end of the channel where an analyte detector is located. The convective flux resulting from the perpendicular cross flow in the channel promotes separation of analytes based on differences in their diffusion coefficients. An analyte's diffusion coefficient is inversely related to its size as defined by the Stokes-Einstein equation. D=kBT/6πnr, where D is the analyte's diffusion coefficient, kB is Boltzmann's Constant, T is the absolute temperature, n is the dynamic viscosity of the liquid, and r is the analyte's hydrodynamic radius (i.e., the analyte's size). Separations based on an analyte's diffusion coefficient are therefore similar to separations based on size.
During FFF separation, the cross flow drives analytes toward the accumulation wall, and the analyte concentration increases with decreasing distance from the accumulation wall. This creates an analyte concentration gradient that triggers the analyte's diffusion away from the accumulation wall and toward the center of the channel, where the flow rate is greatest because of the parabolic flow profile. Therefore, analytes with higher diffusion coefficients (i.e., smaller sizes) will diffuse into areas within the channel where the flow rate is greatest, and will elute earlier than larger analytes. This explanation for FFF separation is valid for biological macromolecules and submicron particles, but is not applicable to larger micron-sized particles.
FFF is a relatively new methodology for size-based separations, especially for the separation of biological macromolecules. However, FFF applications for size-based separations have not attained wide usage due to the requirement for specialized and expensive equipment. Additionally, FFF separations are relatively slow, often requiring an hour or more for completion.
Several known electrophoretic methods are capable of separating analytes based on their size. These methods include gel electrophoresis and capillary electrophoresis.
Gel electrophoresis utilizes an electric field to mobilize analytes through a semisolid medium, most often composed of polyacrylamide or agarose. Gel electrophoretic methods for sizing have mostly been applied to biological molecules such as proteins and nucleic acids. These methods are ubiquitous, but they have several drawbacks. First, analytes are often pre-treated under harsh conditions prior to analysis. In many cases, pre-treatment alters the analytes' structure and size, creating inaccuracies in sizing results. Second, gel electrophoresis is relatively labor intensive. Third, analysis times are lengthy, often in excess of two hours. Fourth, sample throughput is low. Generally only 5-10 samples can be analyzed in a single gel.
Capillary electrophoresis (CE) is an alternative to gel electrophoresis. CE sizing techniques utilize a narrow-bore capillary containing a polymer solution acting as a sieving matrix to which an electric field is applied. Although CE offers a potential for higher sample throughput and faster analysis times than gel electrophoresis, some sizing results in CE are inaccurate due to destructive pretreatment of samples, notably proteins.
Analytical ultracentrifugation (AUC) is a non-chromatographic method that separates analytes of different sizes based on their sedimentation velocity when rotated in a centrifuge. AUC is a low throughput technique, often only able to process a few samples at a time. Each analysis requires several hours. AUC is also a highly specialized technique requiring significant user training prior to routine use.